Many investigators in the selenium field have proposed that selenocompounds are responsible for the many health benefits attributed to selenium, while others have suggested that selenoproteins are likely responsible. These health benefits include preventing cancer, heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, slowing the aging process and having roles in mammalian development, male reproduction and immune function. Our laboratory was the first to propose that these health benefits are due largely to the presence of selenium in selenoproteins as the amino acid, selenocysteine (Sec). To elucidate the role of selenoproteins in cancer prevention and development, we are characterizing the function of several selenoproteins. During the past year, we have focused our attention on studying in greater detail the role of two selenoproteins, thioredoxin peroxidase 1 (TR1) and glutathione peroxidase 4 (GPx4). We had previously shown that the knockdown of TR1 using RNA interference technology in a lung cancer cell line resulted in most of the malignant phenotypes being reversed more towards those of normal cells suggesting that TR1 deficiency is antitumorigenic. This past year, we have focused on the molecular basis of TR1s role in cancer development. We observed that among selenoproteins TR1 is uniquely over-expressed in a number of cancer cell lines and its knockdown in DT cells, a malignant mouse cell line that is driven by k-ras, resulted in morphological changes characteristic of the parental (normal) cells. When these cells were grown in serum-deficient medium, TR1 deficient malignant cells lose self-sufficiency in growth, show defective progression in their S phase and decreased expression in DNA polymerase alpha. These studies provide evidence that TR1 is critical for self-sufficiency in growth signals of malignant cells, that TR1 acts largely to maintain the cancer characteristics of malignant cells and further substantiate that TR1 is a primary target in cancer therapy. In expanding our studies on TR1 knockdown to include a breast cancer cell line as well as the lung cancer cell line, we have found that TR1 deficient cancer cells are more sensitive to TNF-alpha induced apoptosis than control cells and TR1 deficiency is possibly involved in decreased activation of ERK, that invasion and chemotaxis are significantly reduced and that one of the MAP kinases, JNK2, may possibly be involved in invasion and chemotaxis in the breast cancer cell line. As a sidelight to our project on the role of TR1 in cancer, we challenged the cancer stem cell hypothesis which posits that tumor growth is driven by a rare subpopulation of cells, designated cancer stem cells. Studies supporting this theory are based in large part on xenotransplantation experiments wherein human cancer cells are grown in immunocompromised mice and only cancer stem cells, often constituting less than 1% of the malignancy, generate tumors. We used our mouse lung and breast cancer cell lines to show that all colonies derived from randomly chosen single cells in these two malignant cell lines form tumors following allografting histocompatible mice. Our study suggested that the majority of malignant cells rather than cancer stem cells can sustain tumors and that the cancer stem cell theory must be reevaluated. We also examined the intracellular role of GPx4. GPx4 is a selenium-containing, antioxidant enzyme whose intracellular function has not been established. To elucidate its function intracellularly, we knocked down GPx4 in a mouse fibroblast cell line (NIH3T3 cells) that resulted in severe damage to the cells when the level of this enzyme was reduced below 80% of its normal level. Partial reduction in its expression led to morphological changes associated with membrane function. Unexpectedly, GPx4 knockdown cells showed unchanged levels of reactive oxygen species, but these cells manifested highly increased levels of oxidized lipid byproducts. The data thus far have established an essential role of GPx4 in the metabolism of membrane lipid hydroperoxides, and surprisingly, a limited role as a general antioxidant enzyme.